The long-term objective of this research project is to provide a molecular description of the events involved in the regulation of enzyme catalysis. The system under investigation involves the rigorously controlled assimilation of nitrogen in bacterial cells by the enzyme glutamine synthetase (GS). The activity of this enzyme is controlled by transcriptional activation, posttranslational modification and classical allosteric inhibition. An in-depth study is planned of catalysis and regulation of GS and of the enzymes that regulates its activity and genetic expression. The experimental approaches include protein chemistry, enzyme kinetics, spectroscopic techniques (EPR, NMR fluorescence) and site- directed mutagenesis. The specific aims are: 1) to understand the control of the catalytic activity of GS by covalent adenylylation and deadenylylation of tyr-397. This modification affects the conformation of different regions of the protein and couples this regulatory signal to changes in catalytic activity. 2) To identify amino acids at the site surrounding tyr-397 of GS that is recognized by adenylyl transferase (ATase). 3) To understand how the regulatory protein PII interacts with ATase to ultimately control the adenylylation of Gs. 4) To systematically explore the catalytic mechanism of Gs by mutagenesis of amino acid residues considered to be involved in substrate binding, catalysis and regulation. 5) To construct a free-energy profile for the entire catalytic reaction for wild-type and mutant enzymes to aid in identifying the contribution to catalysis of each amino acid residue. 6) To understand the role of the n1 and n2 metal ions in electrophilic catalysis in the formation of the two intermediates, gamma-glu-P and the tetrahedral adduct formed between gamma- glu-P and NH3. 7) To study the proteins involved in the regulation of transcription of GS, viz., NRI, NRII and PII, especially how they interact with each other and with DNA.